Fluorescent in situ hybridisation (FISH) employing nucleic acid probes is one of the most advanced techniques for detection and enumeration of microorganisms. The technique emerged in the early nineties and since then has been improved rapidly and is being used for a wide range of applications which include diagnostics in clinical microbiology and analysis of microbial community structure in environmental and industrial microbiology/biotechnology. Although widely used for bacteria, very few publications describe methods for detection and enumeration of protozoan pathogens. The design of oligonucleotide probes requires skill and experience to determine accessible regions of rRNA in native ribosomes. An additional problem of successful FISH for protozoa is the development of hybridisation protocols that allow oligonucleotide probes to penetrate protozoa cell walls which are fundamentally different to bacterial cell walls. Moreover, the composition of bacterial cell walls has been well documented whereas little knowledge exists about the structure of the cyst walls of protozoa like Cryptosporidium spp, Giardia spp and related organisms.
To date, monoclonal antibodies (mabs) are the most important and widely applied tool for detection of Giardia cysts in water samples. The vast majority of commercially available antibodies show a lack of specificity as the antibodies detect all Giardia spp including species that do not infect humans. As a positive antibody reaction does not allow any conclusion regarding the viability (infectivity) of the cysts, viability stains (DAPI, PI) have to be used in conjunction with antibodies.
Oligonucleotide probes for FISH have several advantages over mabs in that probes are significantly cheaper to produce and are more stable as probes can be. stored for long periods without loosing reactivity or specificity. Furthermore, correctly designed probes should only detect cysts of Giardia lamblia and no other species unable to infect humans. Probes target rRNA and will potentially only detect viable cysts which are able to cause infection. As non-viable (dead) cyst contain no or only small amounts of rRNA, it is envisaged that these cysts will not undergo detection.
The present inventors have developed specific oligonucleotides suitable for detection of potentially viable Giardia spp cysts and hybridisation protocols that allow permeabilization of the cyst walls and enable oligonucleotide probes to reach their ribosomal nucleic acid targets.